Hypoglycemic action of a novel constrained analog of human growth hormone-(6-13).

The amino-terminal region of human GH (hGH), in particular the amino acid sequence Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala[hGH-(6-13)], has been implicated as a functional region for the regulation of energy metabolism by exerting an insulin-potentiating action on insulin-sensitive tissues. Recent structural studies have revealed that the cyclization of the aspartate (Asp11) residue to form the alpha-aminosuccinimide (Asu11) ring is essential for the biological action of peptides related to this hGH fragment. The pharmacological application of these hGH-(6-13) peptides has been hindered by the vulnerability of the alpha-aminosuccinimide to hydrolytic modification leading to the loss of biological action. We have succeeded in stabilizing the structure of the Asu11-hGH-(6-13) peptide by replacing the alpha-aminosuccinimide ring with compatible and less rapidly metabolized gamma-lactam structures. In the present paper we report the bioactivity profile of an analog of hGH-(6-13) containing a gamma-lactam at residue position 11 that mimics the stereoelectronic and conformational characteristics of the alpha-aminosuccinimide ring. In vitro, the gamma-lactam11-hGH-(6-13) peptide analog increased [14C]glucose incorporation into glycogen in muscles and conversion to lipid in adipose tissues. In vivo, the gamma-lactam11-hGH-(6-13) peptide enhanced hypoglycemia during iv insulin tolerance tests. The results demonstrate that the gamma-lactam11-hGH-(6-13) peptide analog has similar biological properties to the Asu11-hGH-(6-13) peptide fragment, but with improved molecular stability and bioavailability.

Synthesis and conformation of constrained peptides with hypoglycaemic activity derived from human growth hormone.

The alpha-aminosuccinimide (Asu11) octapeptide analogue of human growth hormone hGH[6-13] (Leu6-Ser-Arg-Leu-Phe-Asu-Asn-Ala13) has been reported [Robson et al. (1990) Biol. Chem. Hoppe Seyler 371, 423-431] to have hypoglycaemic activity whilst the corresponding peptide with Asp at position 11 is inactive. In order to determine whether this change in activity is caused by conformational and/or stereo-electronic effects, the incorporation of two different isomeric gamma-lactam structures at position 11 has been investigated. One lactam structure (i) is of the type developed by Freidinger and coworkers [Freidinger et al. (1982) J. Org. Chem. 47, 102-107], whilst the isomeric gamma-lactam structure (ii) represents a new type of constrained synthon for use in peptide synthesis. The chiral type-ii gamma-lactam was synthesized via a suitably protected desoxodipeptide prepared in several ways from L-aspartic acid. The solution conformations of the [Asu11]- and the [gamma-lactam11]-containing hGH[6-13] peptide analogues were investigated with the aid of two-dimensional NMR (COSY and NOESY) spectroscopy. Conformational similarities were found for these hGH[6-13] peptide analogues. For example, for all peptide analogues studied, weak NOEs were evident between the Phe10 ring protons and protons of the amino acid residues at the C-terminus. Overall, however, the NOESY NMR spectra of the [Asu11]- and the [gamma-lactam11]-containing peptides related to hGH[6-13] suggest the presence of an extended structure in solution with a possible weak type II' beta-turn at position 11. The extent of conformational constraint introduced into these hGH[6-13] peptide analogues by substitution of the Asu11 residue with either isomeric gamma-lactam structure was reflected as differences in their hypoglycaemic activity. In particular, the hGH[6-13] peptide analogue derived from the new chiral type-ii gamma-lactam exhibits both lower activity in intravenous insulin tolerance tests in vivo and weaker NOEs than the isomeric hGH[6-13] peptide analogue derived from the type (i) gamma-lactam structure. The relative change in blood glucose levels from 20 to 90 min for the racemic (R,S)-form of the type-ii gamma-lactam compared to the control values was approximately half that of the (S)-stereoisomer.

Routine preparation of thiol oligonucleotides: application to the synthesis of oligonucleotide-peptide hybrids.

Oligonucleotide-peptide hybrids have potential for use as antisense inhibitors of gene expression, with the peptide helping to increase the intracellular concentration of the active oligonucleotide. The preparation of such hybrids can be achieved by the coupling of thiol-derivatized oligonucleotides with maleimido-peptides. We have developed reliable methods for preparing 5'-thiol oligonucleotides in good yields using phosphoramidite chemistry and coupling 6-(tritylthio)hexyl phosphoramidite as the 5'-terminal residue. The use of highly pure thiol phosphoramidite as well as a manual iodine treatment after this coupling were found to be important. Oligonucleotide-peptide hybrids were prepared in high yield (85%) by reacting freshly purified 5'-thiol oligonucleotides with peptides derivatized at their N-terminus with a maleimido functionality.

The synthesis of 'difficult' peptides using 2-hydroxy-4-methoxybenzyl or pseudoproline amino acid building blocks: a comparative study.

A comparative study has been undertaken between Hmb-protected amino acid and pseudoproline building block analogues for use in the solid phase synthesis of 'difficult' peptides. Both of these derivatives act by blocking inter- and intramolecular hydrogen bonding, which has been shown to be a major cause of poor synthesis/quality/efficiency. While the two were shown to result in substantial improvements in the purity of crude peptides, pseudoproline incorporation was found to be superior to Hmb backbone protection. This was due to slow and incomplete coupling of the amino acid immediately following the Hmb amino acid.

Monitoring of solid-phase organic synthesis on macroscopic supports by high-resolution magic angle spinning NMR.

In this paper we demonstrate the efficiency of high-resolution magic angle spinning NMR to monitor solid-phase organic chemistry on macroscopic systems such as Synphase lanterns. The use of the LED sequence eliminates the peaks due to the use of protonated solvents and was also sufficient to decrease the signals due to the matrix. As a direct result, we established that reaction kinetics on the lantern proved to be significantly more rapid than on an equivalent polystyrene resin. More generally, the macroscopic nature of the support facilitates both sample preparation and spectral recording and hence opens up the perspective of an automated on-line analysis in combinatorial chemistry.

Synthesis and evaluation of constrained peptide analogues related to the N-terminal region of human growth hormone.

The synthesis and incorporation of two different isomeric gamma-lactam structures into peptide analogues related to hGH [6-13] are described. These peptide analogues and the corresponding aspartimide analogue have been tested for hypoglycemic activity with the intravenous insulin tolerance test. One lactam structure is of the type developed by Freidinger and co-workers, while the isomeric gamma-lactam structure represents a new constrained synthon for use in peptide synthesis. We have found that the hGH [6-13] peptide analogue incorporating the Freidinger lactam was more potent and longer lasting than the aspartimide peptide analogue. The hGH [6-13] peptide analogue incorporating the new gamma-lactam has diminished hypoglycemic activity. The relative biological activities of the three peptide analogues and the possible conformational implications at the physiological site of action are discussed.

1H NMR studies of peptide fragments from the N-terminus of chicken and human transthyretin.

Two synthetic peptides corresponding to N-terminal fragments of human and chicken transthyretin have been synthesized and their structures examined in solution using 1H NMR spectroscopy. Complete sequence-specific assignments obtained for the two peptides are reported together with coupling constant and nuclear Overhauser data. The peptides were found to adopt random-coil conformations in aqueous solution. This is consistent with findings from X-ray structures of the native human transthyretin where the N-terminal region could not be defined, presumably because of conformational disorder.

Identification and synthesis of altered peptides modulating T cell recognition of a synthetic peptide antigen.

In studies of T cell responses to synthetic peptides we have observed agonist and antagonist activities associated with contaminants identified within the parent synthesis. The synthesis of two candidate analogues implied by a peptide contaminant formed during the synthesis of La 51-58 (IMIKFNRL) has been carried out. The peptide contaminant was 17-18 Da smaller than the parent peptide consistent with a modified asparagine residue at position 6 and so we synthesised both an aspartimide and a nitrile analogue, representing cyclisation or dehydration of the asparagine residue. The candidate aspartimide and nitrile analogues both bound empty MHC class I molecules to form allo determinants recognised by monoclonal antibodies. These results demonstrate that altered synthetic peptides can bind class I MHC molecules and prompt caution in the use of synthetic peptides as a source of immunising antigen.

CTL recognition of an altered peptide associated with asparagine bond rearrangement. Implications for immunity and vaccine design.

The extent to which peptides containing chemically and post-translationally modified amino acid side chains are recognized by primed CTL has not been clearly defined. We report on the CTL recognition of a MHC class I-restricted peptide containing a cyclized asparagine (succinimide) residue. This modification of the asparagine side chain is a common intermediate structure during deamidation, isomerization, and bond rearrangements of amide-containing amino acids and also occurs as a side reaction in peptide synthesis. The CTL specifically recognized the succinimide-containing peptide showing only weak cross-reactivity at high concentrations of the parent peptide containing unmodified asparagine. Similarly, CTL raised against the parent peptide did not recognize the succinimide derivative of this peptide. Naturally processed forms of these structures are likely to occur given the importance and frequency of deamidation both in vitro and in vivo. Moreover, since succinimide intermediates of deamidated peptides can occasionally be very stable, these peptides have the potential to act as altered self-Ags with significant implications for autoimmunity. In addition, unwanted and potentially hazardous specificities may be elicited when using synthetic peptides in subunit vaccines in which succinimide residues may form spontaneously during storage or chemical synthesis.

Free radical induced polymerization of synthetic peptides into polymeric immunogens.

Free radical induced polymerization of vinyl monomers such as the acryloyl peptides described here is a facile and rapid reaction used routinely, for example, in the polymerization of acrylamide and bisacrylamide for the assembly of polyacrylamide gels. The technology allows the incorporation of many of the same or different peptide determinants into a single polymer chain. In this study large polymers containing multiple copies of peptides representing T- and B-cell determinants of influenza haemagglutinin were constructed. The determinants retained antigenicity after the polymerization procedure and the polymers were highly immunogenic; the levels of antibody obtained after a single dose of polymeric immunogen were at least as great as those achieved only after repeated doses of the equivalent monomeric peptide. The technology has a wide range of potential applications, not the least significant of which is the construction of designer immunogens for third generation vaccine candidates.

Structure and in vivo activity of hypoglycaemic analogues of human growth hormone (6-13).

A range of peptide analogues related to Asu11-human growth hormone (6-13) have been synthesized and tested for hypoglycaemic activity using in vivo insulin tolerance tests. Utilising an alanine scan procedure and a selective amino acid residue approach these structure-activity studies suggest that residues Phe10, Arg8 and the C-terminal beta-turn structure are important for the expression of biological activity.

Solid phase synthesis of peptide aldehyde protease inhibitors. Probing the proteolytic sites of hepatitis C virus polyprotein.

The solid phase synthesis of a set of peptide aldehydes derived from the NS5A/NS5B junction of hepatitis C virus (HCV) viral polyprotein is demonstrated using an oxazolidine linker and the Multipin method. Deletion of the P6 and P5 residues results in a dramatic loss of inhibitory activity.

Improving the performance of an acid-labile 4-hydroxymethyl phenoxyacetic acid (HMP) linker on resin and SynPhase grafted solid-supports.

A replacement of the acetic acid moiety by valeric acid within the 4-hydroxymethylphenoxyacetic acid (HMP) linker (Sheppard RC, Williams BJ. Acid-labile resin linkage agents for use in solid phase peptide synthesis. Int. J. Peptide Protein Res. 1982; 20: 451-454) significantly improved its performance in terms of loading capacity, yield and purity of the final products. The results indicated the spacer-linker combination and type of solid supports are important factors for solid-phase synthesis.

Avoidance of self-reactivity results in skewed CTL responses to rare components of synthetic immunogens.

In studying the CTL recognition of peptide determinants derived from the nuclear Ag La (SS-B), we observed significant skewing of the response toward rare components present within the immunogen. Thus, priming of naive mouse lymphocytes in vitro with a synthetic H-2Kb-binding peptide comprising human La (hLa) residues 51-58 resulted in class I-restricted cytotoxic T cells that failed to recognize naturally presented hLa 51-58 peptide. Instead, the majority of T hybrids recognized a low abundance (< or = 1%) contaminant present at picomolar concentrations in the original synthesis and identified as a peptide adduct containing N,4-t-butyl asparagine at position 6 of the hLa 51-58 sequence. The preferred T cell recognition of the butyl adduct was not due to increased affinity of this peptide for the H-2Kb molecule or to the antagonism of CTL recognizing the unmodified determinant. Rather, the bias in the immune response appeared to be the result of partial self-tolerance to the homologous mouse La 51-58 determinant, which differs from its human counterpart by only a single amino acid at position 1 (T-->I). Accordingly, the CTL response appeared to be focused on "non-self" ligands present within the synthesis, even though they were present at very low concentrations. These observations have significant implications for the use of synthetic peptide vaccines, especially those designed to manipulate responses to self peptides such as tumor Ags in which self-tolerance may result in unexpected reactivity.

Hemagglutination (fimbriae) and hydrophobicity in adherence of Serratia marcescens to urinary tract epithelium and contact lenses.

The capacity of 59 isolates of Serratia marcescens, obtained from urinary tract infections, wounds, and contact lenses or their paraphernalia, to agglutinate erythrocytes from different animal species was tested. Three main patterns were found: mannose-sensitive agglutination of guinea-pig, fowl or horse erythrocyte; mannose-resistant agglutination of chicken or pigeon erythrocytes alone or in combination with mannose-sensitive agglutination; and no agglutination. Hemagglutination capacity was associated with isolates from urinary tract infection, but not with isolates associated with contact lenses. Adherence to human urinary tract epithelium did not correlate with the hemagglutination patterns nor with the origin of the isolates. Some strains of different hemagglutination pattern were selected for the study of hydrophobicity and adherence to contact lens polymers. Hydrophobicity, as determined by degree of partition in hexadecane and water (BATH-values), correlated neither with degree of adherence to contact lens polymers nor with the hemagglutination pattern. For a representative strain there was an excellent correlation (r2 = 0.98) between adherence and the water content (hydrophobicity) of the lens polymers. These results suggest that, as with tissues, other factors interact with hydrophobicity in causing adherence to plastics.